1. Field of the Invention
The present invention is concerned, in a general manner, with stimulating cells and in particular with selectively activating receptors on the cell surface.
2. Description of the Related Art
It is well known that extracellular signals are transmitted through the plasma membrane by way of receptor proteins which are able to convert the extracellular binding of ligands into an intracellular biochemical event. In this way, cell surface receptors activate intracellular signal pathways which lead to different sites in the cell and induce particular events at these sites.
All cell surface receptors are transmembrane proteins or protein complexes which establish a connection between the inside and the outside of the cell. Many receptors undergo a defined change in the protein conformation when their respective ligand is bound. In the case of some receptor types, this change in conformation leads to an ion channel being opened while, in the case of other receptors, the change in conformation leads to the cytoplasmic region of the receptor being affected in such a way that it can associate with intracellular signal proteins and signal enzymes and activate these proteins and enzymes.
A crucial effect of ligands being bound to receptors is frequently that of multimerizing or crosslinking the receptor and thereby activating the intracellular signal cascade. Such a crosslinking of surface receptors can either be effected by the physiological ligands of the receptors or, for example, be effected in vitro using appropriate crosslinking antibodies.
For example, it has been demonstrated, in the case of lymphocytes, that, while it is not possible to stimulate specific antigen receptors as a result of binding F(ab′) fragments, which possess only one binding site, the receptors form clusters, and the cells are activated, as the result of binding (Fab′)2 antibody fragments, i.e. fragments possessing two binding sites. However, a stronger reaction is achieved if lymphocytes are stimulated by intact antibodies which are bound to cells which carry receptors for the immunoglobulin Fc moiety. In other words, activation takes place when receptors are efficiently crosslinked by being bound to many identical antibody molecules which are provided by other cells which possess Fc receptors for the constant regions of the intact antibodies. The receptors are efficiently crosslinked by the antibodies which are immobilized in this way.
In vitro, such a crosslinking can, on the one hand, be achieved by the constant regions of antibodies which are bound to the receptors being crosslinked by way of protein A or by way of other antibodies which bind specifically to the constant regions of the antibodies which are bound to the receptors.
For example, it is known that the members of the TNF (tumor necrosis factor) family act as trimers and that the ligand induced trimerization of their receptors is the critical event in initiating signal transmission.
Dhein et al., INDUCTION OF APOPTOSIS BY MONOCLONAL ANTIBODY ANTI-APO-1 CLASS SWITCH VARIANTS IS DEPENDENT ON CROSS-LINKING OF APO-1 CELL SURFACE ANTIGENS”, the Journal of Immunology, volume 149, 1992, pages 3166-3173 report that efficient crosslinking of the APO-1 cell surface antigen leads to the induction of apoptosis. They show that, while apoptosis is induced in SKW6.4 cells when anti-APO-1 F(ab′)2 fragments which are crosslinked by way of a sheep anti-mouse antibody bind to the APO-1 receptor, the binding of the F(ab′)2 fragments on their own is insufficient to achieve this. The authors conclude from these results that the bivalency of the antibody, which thus possesses two binding sites for the APO-1 cell surface antigen, is insufficient for inducing apoptosis and that, on the contrary, efficient crosslinking of the APO-1 cell surface antigen is necessary in order to achieve this.
The sequence of the APO-1 antigen, and a monoclonal antibody directed against the APO-1 antigen are described in U.S. Pat. No. 5,891,434. This patent specification mentions that the anti-APO-1 antibodies can be used for treating tumors which the APO-1 antigen, with it furthermore being possible to induce apoptosis in different types of cells.
However, it is known that many cells in the body carry the APO 1 cell surface antigen, which means that administering an anti-APO-1 antibody to a tumor patient would lead not only to an attack on the tumor cells but also, in addition to this, to an attack on other, healthy and perhaps even essential cells which also carry the APO-1 surface antigen.
Against this background, the use of the known anti-APO-1 antibodies for treating tumor patients, for example, is only suitable under certain circumstances.
The TRAIL (TNF-related apoptosis-inducing ligand) receptors R1 and R2 represent another type of death receptor which is activated by crosslinking; see Griffith et al., “Functional Analysis of TRAIL Receptors using Monoclonal Antibodies”, The Journal of Immunology, volume 162, 1999, pages 2597-2605. The authors report that, while all the anti-TRAIL-R2 antibodies, and two of the anti-TRAIL-R1 antibodies, were unable to induce any lysis, or only able to induce minimal lysis, of TRAIL-sensitive melanoma cells when they were added to the cells in solution, these antibodies exhibited an increase in their lytic ability when they were immobilized on a culture plate such that they were able to ensure that the TRAIL-R1 and TRAIL-R2 death receptors were crosslinked.
Antibodies against the death receptors TRAIL-R1 and TRAIL-R2 also act nonspecifically on TRAIL-sensitive cells, which means that they are only of slight therapeutic value.
In addition to this, it is known that what are termed bispecific antibodies, i.e. antibodies which possess a specificity for a tumor-associated antigen and a specificity for a surface antigen on defensive cells of the immune system, such as macrophages, T-lymphocytes or natural killer cells, which cells are activated by way of this binding, can be employed in cancer immunotherapy for directing the activity of the defensive cells toward the particular target cells.
In a general manner, bispecific antibodies are antibodies which are able to bind two different epitopes and are monovalent for each epitope. They are prepared by oxidizing monovalent F(ab′) fragments to give an F(ab′)2 fragment, by fusing two hybridoma cell lines to give hybrid hybridoma or quadroma cells, or recombinantly.
Jung et al., “Target cell-induced T cell activation with bi- and trispecific antibody fragments”, Eur. J. Immunol., volume 21, 1991, pages 2431-2435 describe the preparation of bispecific F(ab) hybrid fragments which are monovalent for each antigen. The reader is referred to this publication for further references to the preparation of bispecific antibodies.
The authors demonstrate that bispecific antibodies or fragments can be used to effect a target cell induced activation of T cells, by the antibodies on the one hand binding to the target antigen on the target cell and, on the other hand, binding to the CD3 and/or CD28 receptor on the T cell.
Segal et al., “Bispecific antibodies in cancer therapy”, Current Opinion in Immunology, volume 11, 1999, pages 558-562 also describe the use of bispecific antibodies for directing an effector cell to a target cell which it would not otherwise recognize. For this purpose, the bispecific antibodies bind to a surface molecule on the target cell and to a surface receptor on the effector cell.
Roosnek et al., “T cell activation by a bispecific anti-CD3/anti-major histocompatibility complex class I antibody”, Eur. J. Immunol., volume 20, 1990, pages 1393-1396 showed that a bispecific antibody which possessed specificity both for MHC and for CD3, both of which were expressed on T cells, was able to induce efficient proliferation of T cells whereas a mixture of the two original antibodies was unable to do this. The authors hypothesize that this synergistic effect is due to the anchoring of the T cell receptor/CD3 complex in the membrane being disturbed. In this connection, they make the assumption that the T cell receptor is unable to distinguish whether it is anchored to an antigen-presenting cell (APC) or to surface molecules within its own membrane. They therefore speculate that the T cell receptor/CD3 complex, which in vivo is triggered by antigens on another cell, reacts to changes in its mobility within the membrane.
However, the mechanism of this T cell receptor activation, which is restricted to certain T cell subpopulations, has remained unclear. The prior art has thus far assumed that this is a T cell receptor-specific effect which is probably due to the fact that, in addition to the T cell receptor, a coreceptor such as CD4 or CD8 is stimulated, with this coreceptor also generating a signal on stimulation, see Emmrich et al., Eur. J. Immunol., 18, 645 (1988).